Anatomic localization of B cells within the spleen is associated with different functions in an immune response, but the biochemical pathways that control these differences are unknown. In mice transgenic for IgM carrying the VH81X variable region, B cells expressing an autoreactive idiotype are positively selected and persist in a particular anatomic location in the spleen, but antibody production by these cells is undetectable. In preliminary studies of the biochemical mechanisms of regulation of these autoreactive cells, evidence of increased signaling after antigen receptor (AgR) ligation was observed, in contrast to the suppressed signaling demonstrated in previous transgenic models of B cell clonal anergy. Further data suggest that the increased AgR signaling may be a general property of cells in the same region. this suggests a novel mechanism of regulation of certain auto-reactive B cells in the spleen, associated with increased AgR signaling but suppression of differentiation into antibody secreting cells, likely associated with specialized functions, perhaps related to immunologic memory. To begin to characterize regulatory mechanisms in such cells, this project will analyze 1) differences in AgR signaling in different splenic B cell subsets, 2) differences in AgR signaling between the transgenic auto-reactive cells and other B cells, and 3) the role of accessory membrane proteins in modulating AgR signaling in different subsets and in auto-reactive cells. Changes in intracellular calcium, by flow cytometry, and protein tyrosine phosphorylation, by immunoblotting of whole cell lysates, will be analyzed after AgR ligation in B cells separated into subsets on the basis of cell surface markers. The transgenic auto-reactive B cells will be selectively stimulated with anti-idiotypic or anti-allotypic antibodies and signaling compared to normal cells. Accessory membrane protein function will be compared between normal subsets and between normal cells and those expressing the auto-reactive idiotype. The intermediate goal is to identify cytoplasmic enzymatic pathways, by in vitro reactions, or nuclear transcription factors, using gel shift assays or polymerase chain reaction, that are differentially regulated in B cell subsets, particularly those where auto-reactive cells persist. The long term goal is to find how antibody secretion is regulated in clonally persistent auto-reactive B cells, and to identify potential targets for therapeutic intervention when this regulation fails.